Preparation of single myofiber with satellite cell
Material
- Mouse
- DMEM/PS
- Collagenase I sol (2 mg/ml collagenase I in DMEM/PS, -20C)
- Chicken embryonic extract (CEE) (100% stock in PBS filtrated by 0.2 um filter, -20C) (GEMINI 100-163P, powder, 4C)
- Myofiber medium (DMEM/20% FBS/2% CEE)
- Horse serum (HS)
- Plastic pipet
Protocol
- Euthanize mouse.
- Dissect EDL or TA muscle.
- Wash muscle by 1 ml DMEM/PS in 24-well plate.
- Incubate muscle in 1 ml collagenase I sol in 24 well-plate at 37C for 1 h (EDL) - 2 h (TA).
- Prepare HS-coated 3 cm and 10 cm dish.
- Move muscle to HS-coated 10 cm dish by plastic pipet.
- Dissociate muslce by plastic pipet WITHOUT TOUCHING MUSCLE.
- FOR FLOATING CULTURE: Move single myofiber by 200 ul pipet into myofiber medium in HS-coated 3 cm dish.
- FOR IMMUNOSTAINING: Move single myofiber by 200 ul pipet into 1 ml 2% PFA/PBS in 48-well plate.
See Immunostaining for myofiber.
HS-coated dish
HS-coating prevent myofiber from attaching to dish.
- Coat 3 cm and 10 cm petri dish with horse serum.
- Incubate at 37C for 30 min.
- Remove horse serum.
- Add 1.5 ml myofiber medium into 3 cm dish.
- Add 10 ml DMEM/PS into 10 cm dish.
- Incubate dish at 37C until use.
Reference
Pasut A, Jones AE, Rudnicki MA. Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle. J Vis Exp. 2013; : e50074.